Figure 1
Reaction products of isopeptidase activity. Reaction mixtures for isopeptidase activity contained in a final volume of 50 μl 50 mM Tris/HCl, 1 mM DTE, 5 mM MgCl2, 1 mM ATP, 1 mM EGTA, 0.2 mg/ml first order and second order ubiquityl-calmodulin conjugates and the indicated amounts of affinity-purified isopeptidase activity. Incubation was stopped after 60 min by adding 60 μl of Laemmli sample buffer containing 10 mM EGTA and heating to 96°C for 5 min. The reaction mixtures were analyzed on a 12.5% Weber-Osborne gel. Lane 1: without added isopeptidase activity. Lane 2: with 0.12 mg/ml affinity-purified isopeptidase. Lane 3: with 0.23 mg/ml affinity-purified isopeptidase. Lane 4: with 0.46 mg/ml affinity-purified isopeptidase.